Foreword

The study of ribozymes, RNA-based catalysts, and subsequently nucleic acid-based catalysis has been immensely instrumental in the push of efforts toward a deeper understanding of RNA structure and function. The field attracted many scientists trained in chemistry, biology, or computer science. In 1993, a few years after the Noble Prize in Chemistry (https://www.nobelprize.org/prizes/lists/all-nobel-prizes-in-chemistry) was awarded to Tom Cech [1] and Sidney Altman [2] for the discovery of ribozymes, the RNA Society (www.rnasociety.org) was founded and two years later the RNA Journal (https://rnajournal.cshlp.org) started. Ever since, the annual RNA meetings have gathered more than 1000 scientists from all over the world. Many new techniques and approaches were developed accelerating the pace of discoveries on RNA. For many years, the meetings started with a session on "RNA Catalysis." However, with the avalanche of new data, new RNAs, and new biology, the session on catalysis session has dwindled. Billions of years ago, RNA did start the chemistry of the game of life, but was overwhelmed by the initiated evolutionary processes.

The central group of the chemical and biological feats achieved by RNA is the ribose hydroxyl O2´, at the same time key actor and Achilles's heel. And a DNA phosphodiester bond is 104-105 less prone to cleavage than a RNA phosphodiester bond [3]. As Paracelsus wrote several centuries ago: "All things are poison and nothing is without poison; only the dose makes a thing not a poison".

The catalytic power of the 2´-hydroxyl group must be strongly controlled and amplified only at specific location in the RNA sequence. Catalysis generally is initiated by the formation of an anionic O2´ oxygen. In any biological system, catalysis requires accessibility, local molecular dynamics and solvent molecules (water, ions, or small ligands). A water molecule (or a hydroxide ion), or a solvated divalent ion, or an amine group from a ligand can thus capture the proton from the 2´-hydroxyl group. Afterwards, depending on the available mobility of the nucleotide, the anionic O2´ oxygen can attack its own 3´-phosphate group or another phosphodiester bond. Other types of chemical reactions have also been achieved using ribozymes as described in several chapters of this book.

Accessibility and local dynamics depend on the RNA sequence and the ensuing RNA architecture. The distinctive mark of nucleic acids is the formation of pairs between the bases of the nucleotides with the complementary Watson-Crick pairs the most frequent ones. However, such pairs form only regular helices and any complex fold or assemblies of helices require linking segments that engage in some type of non-Watson-Crick base pairs. Depending on the complexity and compactness of the RNA fold, not many bases will remain unpaired with a large number of degrees of freedom. Such regions are particularly prone to phosphodiester cleavages through hydrolysis or metal ion attack. Further, it was noticed a long time ago that, in single-stranded regions, dinucleotide steps with sequence pyrimidine-adenosine (UpA and CpA) were particularly sensitive to cleavage [4, 5]. These early observations were later thoroughly studied [6, 7]. Such cleavages are regularly observed in control lanes during gel electrophoresis. The precise molecular mechanism for these spontaneous cleavages in YpA sequences, however, has remained elusive. Recently, a surprising interpretation, based on mass spectrometry data and implying the syn conformation of the A and its protonation at N3 position, has been put forward [8].

In each ribozyme, cleavage occurs at a very precise dinucleotide location and generally with weak sequence dependence. The more extensively studied are the endonucleolytic, ribozymes (see Chapters 1 and 2). New data and results confirm the involvement of unexpected chemistry with anionic guanosine residues acting as a general base for capturing the O2´-hydroxyl proton [9, 10]. Interestingly, a proposal has been forwarded in which the tautomeric enol form of guanosine in which N1(G) carries an in-plane electron doublet would capture the proton from the O2´-hydroxyl group [11, 12]. Interestingly, such tautomers have been observed in functional ribosome crystals and related to the occurrence of miscoding at the first and second codon positions following the formation of tautomeric GoU pairs [13, 14]. The tautomeric ratio of G is around 1 in 104, a value close to the average miscoding error in bacteria [15]. The tautomeric forms trapped in a constrained environment (stacking, H-bond between the amino group of G and an anionic phosphate oxygen, minor groove contacts, etc.) are stabilized as observed in several crystal structures [12, 16]. The frequent occurrence of stacked Gs forming H-bonds through their Watson-Crick edge to phosphate anionic oxygens has been analyzed and described thoroughly [17]. It has also been shown that the tautomeric form coexists with the anionic form [18].

Overall, this book is timely and unique in its breadth of content. The book describes the great diversity of nucleic acid base catalysis beautifully. The reader is conveyed to a chemical journey extending from biologically functional ribozymes (like RNaseP or group I and II introns) to engineered and designed ribozymes as well as DNAzymes. For example, we now have three distinct structural environments with a natural 2´-5´ phosphodiester linkage: the group II ribozyme [19, 20], the spliceosome [21], and the lariat-capping ribozyme [22]. Despite the clear structural similarities between the active sites of group II ribozyme and of the spliceosome, the striking observation is how the 2´-5´ linkage promotes a highly constrained environment with nested non-Watson-Crick pairs and sugar-phosphate contacts. Given those three-dimensional visions, one can only wonder at the structural and molecular fitness of nucleic acids, especially RNA. Here I have selected some topics that I enjoy particularly, and I apologize for not discussing many other aspects that are covered in this book. Indeed, some chapters extend to biotechnology and to ribozymes used in diagnostics and therapeutics. Finally, the major tools and techniques used for the analysis of nucleic acid structures are presented up to date. In this regard, I cannot resist citing Sydney Brenner [23, 24], Nobel Prize 2002 in Physiology or Medicine: "Progress in science depends on new techniques, new discoveries and new ideas, probably in that order."

Eric Westhof

Architecture et Réactivité de l´ARN

Université de Strasbourg

Institut de biologie moléculaire et cellulaire du CNRS

2 allée Konrad Roentgen, 67084 Strasbourg, France

August 2019

References

1. 1 Cech, T.R., Zaug, A.J., and Grabowski, P.J. (1981). In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell 27 (3 Pt 2): 487-496.
2. 2 Guerrier-Takada, C., Gardiner, K., Marsh, T. et al. (1983). The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35 (3 Pt 2): 849-857.
3. 3 Thompson, J.E., Kutateladze, T.G., Schuster, M.C. et al. (1995). Limits to catalysis by ribonuclease A. Bioorg. Chem. 23 (4): 471-481.
4. 4 Cannistraro, V.J., Subbarao, M.N., and Kennell, D. (1986). Specific endonucleolytic cleavage sites for decay of Escherichia coli mRNA. J. Mol. Biol. 192 (2): 257-274.
5. 5 Kierzek, R. (1992). Hydrolysis of oligoribonucleotides: influence of sequence and length. Nucleic Acids Res. 20 (19): 5073-5077.
6. 6 Kaukinen, U., Lyytikainen, S., Mikkola, S., and Lonnberg, H. (2002). The reactivity of phosphodiester bonds within linear single-stranded oligoribonucleotides is strongly dependent on the base sequence. Nucleic Acids Res. 30 (2): 468-474.
7. 7 Soukup, G.A. and Breaker, R.R. (1999). Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5 (10): 1308-1325.
8. 8 Fuchs, E., Falschlunger, C., Micura, R., and Breuker, K. (2019). The effect of adenine protonation on RNA phosphodiester backbone bond cleavage elucidated by deaza-nucleobase modifications and mass spectrometry. Nucleic Acids Res. 47(14): 7223-7234.
9. 9 Wilson, T.J., Liu, Y., Li, N.S. et al. (2019). Comparison of the structures and mechanisms of the pistol and hammerhead ribozymes. J. Am. Chem. Soc. 141 (19): 7865-7875.
10. 10 Bevilacqua, P.C. (2003). Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. Biochemistry 42 (8): 2259-2265.
11. 11 Pinard, R., Hampel, K.J., Heckman, J.E. et al. (2001). Functional involvement of G8 in the hairpin ribozyme cleavage mechanism. EMBO J. 20 (22): 6434-6442.
12. 12 Singh, V., Fedeles, B.I., and Essigmann, J.M. (2015). Role of tautomerism in RNA biochemistry. RNA 21 (1):...